International Journal of Research in Engineering and Science (IJRES) ISSN (Online): 2320-9364, ISSN (Print): 2320-9356 www.ijres.org Volume 2 Issue 5 ǁ May. 2014 ǁ PP.49-54 www.ijres.org 49 | Page Effects of Nitrogen Concentration and Culturing Temperatureon Lipase Secretion and Morphology of the Antarctic Basidiomycetous Yeast Mrakiablollopis Masaharu Tsuji 1 , Yuji Yokota 2 , Sakae Kudoh 3 , Tamotsu Hoshino 1 1 Biomass Refinery Research Center (BRRC), National Institute of Advanced Industrial Science and Technology (AIST), Hiroshima, Japan 2 Bio-production Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Hokkaido, Japan 3 National Institute of Polar Research (NIPR), Tokyo, Japan ABSTRACT :The effect of nutrient concentration on Mrakiablollopis SK-4 colony morphology and the effects of nitrogen concentration on formation of a clear zone around colonies, which is indicative of lipase activity, and on morphology were examined on PDA and fresh cream agar at various culturing temperatures. When the yeast was inoculated on a eutrophic medium, it maintained its yeast form, while it showed an almost mycelial form on an oligotrophic medium regardless of culturing temperature. When grown on high-nitrogen fresh cream agar, the largest clear zone was formed around colonies at 4°C and the morphology was considered to be a yeast form. Morphology of SK-4 was changed by the nutrient condition under the colony on an agar plate. Secretion of lipase was increased by a high nitrogen concentration. SK-4 is thought to take the yeast form in aquatic environments, and this form may secrete more lipase than the mycelium form. Keywords -Cold-adapted yeast• Cryophilic fungi• Fluorescence in situ hybridization• Lipolytic enzyme• Mrakia I. INTRODUCTION Lipase is known as one of the most important enzyme for industries. Cryophilic fungi [1] have been reported worldwide in cold environments including the polar regions. The cryophilic basidiomycetous yeast Mrakia spp. and Mrakiella spp. have been found in areas such as Antarctica, the Alps [2], Central Russia [3], Siberia and the Arctic [4]. diMenna [5] reported that Mrakia spp. accounted for about 24% of the culturable yeast in Antarctic soil. Furthermore, Fujiu reported that about 25% of culturable fungi isolated from lake sediments and soils of East Antarctica were Mrakia spp. (Master's thesis, Graduate School of Science, Hokkaido University, 2010). These reports indicated that Mrakia spp. are the dominant fungi in Antarctica and the most adaptive to the Antarctic environment. The cryophilic yeast M. blollopis SK-4 was isolated from an algal mat from the oligotrophic lake Naga- ike, Skarvsnes ice-free area, East Antarctica [6]. When SK-4 was inoculated on agar plates containing fresh cream as the carbon source, this yeast formed the largest clear zone, formed by lipase activity, at 10°C [7]. However, there is little known about how SK-4 lipase production is influenced by nitrogen concentration, culturing temperatures or cell morphology. Moreover, there is little known about how Mrakia spp. lives under such oligotrophic conditions. Here, we report on the effects of nutrient concentration on SK-4 cell morphology, and the effects of nitrogen concentration on lipase secretion and cell morphology at different culturing temperatures. We also observed cell morphology on lipase production medium using fluorescence in situ hybridization (FISH). II. MATERIALS AND METHODS 2.1. Media composition Fresh cream agar (FCA) was composed of 3.2 g/l yeast extract (yeast extract containing 109 mg/g nitrogen and 163.3 mg/g carbohydrate), 5.4 g/l peptone (peptone consisting of 154 mg/g nitrogen and 13.3 mg/g carbohydrate), 5.4 g/l NaCl, 55 ml/l fresh cream (fresh cream containing 466 mg/ml milk fat and 23.5 mg/ml carbohydrate) and 20 g/l agar, pH 7.0. 2× FCA was identical to FCA except for 10.8 g/l peptone, and 5× FCA was identical to FCA except for 27.0 g/l peptone. 1/5 potato dextrose agar (PDA) was composed of 7.8 g/l PDA (DB Japan, Tokyo, Japan) and 14 g/l agar. 2× PDA was composed of 39 g/l PDA and 24 g/l potato dextrose